Method of synthesizing peptides in the presence of a carbodiimide and a 1-hydroxy-benzotriazole

ABSTRACT

Improved synthesis of peptides by the carbodiimide method in which an amino-protected amino acid or peptide having a reactive carboxy group is condensed with a carboxy-protected amino acid or peptide having a reactive amino group in the presence of a 1hydroxy-benzotriazole or a substituted 1-hydroxy-benzotriazole of the formula   AS WELL AS IN THE PRESENCE OF A CARBODIIMIDE SUCH AS DICYCLOHEXYL CARBODIIMIDE.

United States Patent 1 Kiinig et al.

[54] METHOD OF SYNTHESIZING PEPTIDES IN THE PRESENCE OF A CARBODIIMIDEAND A l-HYDROXY- BENZOTRIAZOLE 1 [75] Inventors: Wolfgang Kiinig,Langenhain/Taunus; Rolf Geiger, Frankfurt/Main, both of Germany [73]Assignee: Farbwerke Hoeclist Aktiengesellschait vormals Meister, Lucius& Bruning, Frankfurt am Main, Germany 1 Filed: Apr. 1, 1970 Appl. No.:24,844

[30] Foreign Application Priority Data FOREIGN PATENTS OR APPLICATIONS1,073,500 l/1960 Germany ......260/112.5

[ 5] Apr. 3, 1973 OTHER PUBLICATIONS Anderson et al., Actu. Chem. Acad.Sci. Hung 44, 51-59(1965). Zimmerman et al., J. Amer. 7,151-7,152(1967). Paquette, J. Am. Chem. Soc. 87, 5,l865,190 (1965). Singh et al.,J. Org. Chem. 25, 657-658 (1960). Konig et al., Chem. Ber. 103, 788798(1970).

Chem. Soc. 89,

Primary Examiner-Elbert L. Roberts Attorney-Curtis, Morris & Safford[57] ABSTRACT Improved synthesis of peptides by the carbodiimide methodin which an amino-protected amino acid or peptide having a reactivecarboxy group is condensed with a carboxy-protected amino acid orpeptide having a reactive amino group in the presence of a 1-hydroxy-benzotriazole or a substituted l-hydroxybenzotriazole of theformula as well as in the presence of a carbodiimide such asdicyclohexyl carbodiimide.

5 Claims, No Drawings METHOD OF SYNTHESIZING PEPTIDES IN THE PRESENCE OFA CARBODIIMIDE AND A 1- HYDROXY-BENZOTRIAZOLE The present inventionrelates to a method for synthesizing peptides in the presence of acarbodiimide and a l-hydroxy-benzotriazole.

One of the easiest process for the preparation of peptides consists inlinking a N-acyl-amino acid or a N- acyl-peptide with an amino acidester or peptide ester by means of dicyclohexyl-carbodiimide [.l. C.Sheehan and G. P. Hess, J. Am. Chem. Soc. 77, 1,067 (1955)]. This methodhas the disadvantage of a quite considerable racemization in the linkingof the peptides [E Weygand, A. Prox and W. Kbnig, Chem. Ber. 99,1,4511,46O (1966)] and the formation of N-acylureas, which make theproduct of the synthesis impure and reduce the yield. (Cf. E. Schr6derand K. Liibke, The Peptides, Vol. 1., pages 108 lll, Academic Press, NewYork and London 1965).

Additions of 1.1 to 2 equivalents of N-hydroxy-succinimide duringpeptide synthesis with dicyclohexylcarbodiimide reduce the racemizationto below 1 percent of D-compound and prevent the formation of N-acyl-ureas [F. Weygand, D. Hoffmann, E. Wiinsch, Z. Naturf. 21 b,426-428 (1966); J. E. Zimmermann and G. W. Anderson,J. Am. Chem. Soc.89, 7,151 (1967)]. The addition of N-hydroxy-succinimide in thedicyclohexyl-carbodiimide method, however, has serious disadvantages. Itis reported in Acta Chim. Hung. 44, 63-65 (1965) thatN-hydroxy-succinimide itself reacts with dicyclohexyl-carbodiimide andthat sterigeneral formula I amino-acid that may be protected or apeptide that may be protected or with the amides thereof, the aminogroup of said components being free, in one of the solvents usuallyemployed in peptide chemistry. After the cally hinderedN-acyl-peptide-N-hydroxy-succinimide esters could not be prepared. Acompound formed from one mol of dicyclohexyl-carbodiimide with threemols of N-hydroxy-succinimide was later on identified assuccinimidooxycarbonyl-B-alanine-hydroxy-succinimide ester (E. Bricas:Peptides, North Holland Publishing Comp. (1968), pages 156 157).

This compound reacts smoothly with amines to yield urea-derivatives ofthe B-alanine-amides [cf. also Z. Naturf. 23 b, 1,391 (1968)]. Afterreaction of BOC-L- glutamic acid-a-benzyl ester withN-hydroxy-succinimide and dicyclohexyl-carbodiimide and followingaddition of 2,4,6-trimethoxy-benzylamine, a considerable amount ofsuccinimido-oxycarbonyl-B-alanine-2,4,6- trimethoxy-benzylamide wasisolated.

Now, we have found that, surprisingly, in the dicyclohexyl-carbodiimidemethod the addition of lhydroxy-benzo-triazoles (HOT) can also reduceracemization in the racemization test according to Chem. Ber. 99,1,45l1,460 (1966), to below 1 percent of D-compound and preventsN-acyl-urea formation (cf. Table 1 in Example 1, 1). In additionthereto, no byproducts can be formed, as those which can be formed uponaddition of N-hydroxy-succinimide. The melting points and yieldsindicated in Table 3 show that in the peptide synthesis with DDC thel-hydroxybenzotriazoles are distinctly superior to known addition ofN-hydroxy-succinimide (tests 26 29).

Hence, the object of the present invention is a process for themanufacture of peptides wherein a protected amino-acid or a protectedpeptide, in which the carboxyl group which shall enter into reaction isfree, is reacted with a protected amino-acid or peptide ester or amide,in which the amino group which shall enter into reaction isfree, in oneof the solvents usual in peptide chemistry with the addition of acarbodiimide and of 1 2 equivalents of a l-hydroxy-benzotriazole of thereaction is complete and the product is purified, the protective groupsare split off completely or partially, if desired or required.

If n 1, R may also stand for different substituents.

From N-protected amino-acids and peptides, there can be prepared with'1-hydroxy-benzotriazoles and dicyclohexyl-carbodiimide activatedproducts which are excellently suitable for peptide synthesis. Thereby,activated esters are formed which, in solution, stand in an equilibriumwith an activated amide. Depending on the substance, either theactivated ester or the activated amide can be isolated. In general,however, isolation of the activated product is not necessary.

Dicyclohexyl carbodiimide R N O R'COOH stands for N-protectedamino-acids and N- protected peptides.

These new activated compounds react very rapidly usingN-hydroxy-succinimide ester, the percent stage is reached only after 2hours at 0 C. A further advantage is that the esters need not beisolated, because l-hydroxy-benzotriazole and its derivatives do notform by-products with dicyclohexyl-carbodiimide, in contradistinction toN-hydroxy-succinimide. This can be proved very easily by paperchromatography, for example on a partially protectedcorticotropin-(ll-23)- tridecapeptide-amide having a free a-amino group,described in Chem. Ber' 97, 1,207 (1964). If this compound is storedwith dicyclohexyl-carbodiimide and hydroxy-succinimide indimethylformamide or a similar solvent, it can after some hours nolonger be detected by chromatography because it has reacted to formother compounds. With dicyclohexyl-carbodiimide andl-hydroxy-succinimide, the trideca-peptide was unchangedeven afterhours.

In the activation of N-protected peptides which do not contain prolineand glycine as the amino-acid at the carboxylic end group, a slightracemization is to be expected. But even N-hydroxy-succinimide esterscannot be prepared completely free of racemization withdicyclohexyl-carbodiimide (cf. Table 2, Example I, 2). In addition tol-hydroxy-benzotriazole itself, the following derivatives may also beused: 6-chloro-1- hydroxy-S-isopropyl-benzotriazole, orS-chloro-lhydroxy-7-methyl-6'nitro-benzotriazole, as well as thecompounds mentioned in the examples.

As carbodiimides, the compounds usually employed in peptide chemistry,such as N,N-dicyclohexyl-carbodiimide, N,N'-di-isopropyl-carbodiimideand watersoluble carbo-diimides, may be used. As protective groups forthe functional groups of the amino-acids and peptides which have to beprotected, all the protective groups usually employed in peptidechemistry are suitable. Even polymeric resins, for examplehydroxymethyl-polystyrene, may be used as protective groups [E.Schriider, K. Liibke, The Peptides, Vol. I, pages 108-111, AcademicPress, New York and London For preparing the l-hydroxy-benzotriazolessubstituted at the nucleus which are used according to the invention andwhich have not yet been described in the literature,o-chloro-nitrobenzenes of the general formula II, in which R and n havethe meanings given above, are heated with 3 equivalents ofhydrazinehydrate or with 1 equivalent of hydrazine-hydrate and 2equivalents of triethylamine in alcohol and the hydrazineandtriethylamine-salt, respectively, of the l-hydroxy-benzotriazole whichis formed is dissolved in water and acidified, whereupon the clearlysubstituted l-hydroxy-benzotriazole precipitates.

In the case of carboxyl-protected peptides which are sparingly solublein water, the l-hydroxy-benzotriazole added can be completely extractedby shaking with sodiumor potassium-bicarbonate solution or with a sodasolution. A particular advantage of this method is that thel-hydroxy-benzotriazole used can be again precipitated by acidificationof these sodium bicarbonate washing solutions. This is not possible ifN- hydroxy-succinimide is added, for example, since this compound alsodissolves in acid solutions. l-hydroxybenzotriazole can be extractedfrom sparingly-soluble peptides with isopropanol, alcohol, methanol,tetrahydrofurane or hot water.

In all the examples tested, the yield obtained with the addition ofl-hydroxy-benzotriazoles was higher than with or without addition ofN-hydroxy-succinimide; for example, a protected decapeptide having thesequence of the antamanide, a cyclodecapeptide from Amanita phalloides,could be prepared according to the dicyclohexyl-carbodiimide method fromZ-Phe-Phe- Pro-Pro-OH and H-Phe-Phe-Val-Pro-Pro-Ala-OBu with theaddition of l-hydroxy-benzotriazole in a yield of 91.4 percent. Upon acorresponding addition of N- hydroxy-succinimide, a yield of only 61percent was obtained.

The new method is also successful in the preparation of corticotropin-(1-23 )-tricosapeptide-amide as described in GermanPatent No. 1,240,088.There, 4- nitrophenol is added to the dicyclohexyl-carbodiimide.However, pentachlorophenol or hydroxyl-succinimide are better than thisadditive. When taking the yields obtained with the latter additives as100, then the yield without addition is 60, with nitrophenol it is 80,but with l-hydroxy-benzotriazole it is 140, as has been found bybiological assay and by a method of quantitative determination usingphotometric evaluation of the color of the ninhydrine-cadmium complexafter paper chromatographic separation of the crude product in a manneranalogous to the process described in Naturwiss. 42, 416 1955).

Table 1 shows that racemization in the dicyclohexylcarbodiimide methodin tetrahydrofurane as the solvent is reduced to below 1 percent if 2equivalents of 1- hydroxy-benzotriazole are added. If dimethylformamideis used as the solvent, a slight racemization takes place upon additionof 2 equivalents of unsubstituted lhydroxybenzotriazole, while with anaddition of 1 1.2 equivalent racemization remains below 1 percent. It isquite the reverse if N-hydroxy-succinimide is used as an additive. If apeptide is activated by means of dicyclohexyl-carbodiimide andl-hydroxybenzotriazole, the degree of racemization is the lowest evenhere with 1 equivalent of l-hydroxybenzotriazole. In tetrahydrofurane,it even remains below 1 percent. If N-hydroxy-succinimide is added, thecourse is here again reversed (cf. Table 2). As is shown in Examples Ill2 and 3 (synthesis of C- GlN(Bz)-OBz), the yields are smaller when 2equivalents of N-hydroxy-succinimide are added rather than 1.equivalent, and the reaction products are considerably less pure. Thismeans that the low degree of racemization upon addition ofN-hydroxy-succinimide is purchased with the formation of manyby-products.

If, however, peptides are synthesized according to the one-pot method byreaction of the carboxyl component with the amino component by means ofa carbodiimide with addition of a l-hydroxy-benzotriazole of the generalformula I, it is suitable to use 2 equivalents of the correspondingl-hydroxybenzotriazole of the general formula I.

In some cases the new activated esters were isolated. Serine peptidescan be prepared with excellent yields according to the one-pot method.For example, the Z- Ser.Gly-ONB could be prepared in a yield of 97.5percent according to the dicyclohexyl-carbodiimide method with additionof 2 equivalents of I-hydroxybenzotriazole. Without the addition ofl-hydroxybenzotriazole, the yield was only 70 percent [Helv. Chim. Acta42, 1,702 (1959)].

Accordingly, the new process has great importance for the manufacture ofserine peptides because the azide method used generally gives loweryields. Moreover, 4-carbobenzoxy-aminoxazolidinone-2 is formed as abyproduct from N-carbobenzoxy-serine azide by way of the isocyanate. Ifdicyclohexyl-carbodiimide is used alone, N-acyl-urea is formed asbyproduct [E. Schriider and K. Liibke, The Peptides, Vol. I, page 208,Academic Press, New York and London (1965)].

If glutaminylor asparaginyl-peptides are prepared with the aid ofdicyclohexyl-carbodiimide, corresponding nitriles are formed inconsiderable amounts by dehydration of the acid amide group. WithN-acylasparagin, an imide formation is also possible. Thus,asparaginyl-peptides can only be prepared with yields of 39 45 percent[E. Schroder and K. Liibke, The Peptides, Vol. I, pages 191, 202-204,Academic Press, New York and London, (1965)]. When, however, 1-hydroxy-benzotriazole is added in the dicyclohexyl-carbodiimide method,glutaminyland asparaginyl-peptides can be prepared with good yields andhigh purity and in a rapid manner. For example, Z-AsN-Leu-OCl-I wasprepared according to the new method in a yield of 85 percent andZ-GlN-Ala-OBu was obtained in a yield of 73.7 percent.

Arginine peptides having a protonized guanido group can also be preparedaccording to the new method with better yield and in pure form. Even thepreparation of the activated ester from protonized N-acylarginine andl-hydroxy-benzotriazole 'is possible, which ester is further reactedwithout isolation.

As components of the peptides prepared according to the process of theinvention, there may be used all the amino-acids in their L- or D-formcontained in naturally occurring peptides. B-Amino-acids, for exampleB-alanine or other, only synthetically or semisynthetically accessibleamino-acids, for example amethyl-alanine,a-methyl-3,4-dioxy-L-phenyl-alanine or B-chloroalanine, may also beused.

After splitting off the protective groups, the products of the inventionmay be used as medicaments or as intermediate products in themanufacture of other therapeutically valuable peptides, such, forexample, as oxytocin, vasopressin, glucagon, ACTH, secretin,thyrocalcitonin, or insulin.

In the present specification and in the examples, the amino-acids areabbreviated in accordance with the in-, ternationally accepted rules. Inaddition, the following abbreviations are used:

AsN asparagine GIN glutamine Z carbobenzoxy- Boc tert.-butyloxycarbonylNPS o-nitrophenyl-sulfenyl- OCH methyl ester ONB p-nitrobenzyl ester Allthe amino-acids mentioned hereafter are used in their L-form.

The following examples illustrate the invention EXAMPLES I.Gas-chromatographic racemization test according to F. Weygand et al.[Chem. Ber. 99, 1,45 l-l,460 (1 966)] TlTe test was modified in thatinstead of Z-Leu-Phe-OH, BOC-Leu-Phe-OH was used,: which has theadvantage that the completely protected peptide BOC-Leu-L.D-Phe-Val-OBuwhich is formed can be hydrolyzed without previous cleavage of theprotective groups.

1. Test for racemization in the dicyclohexyl-carbodiimide method withdifferent additives 378.5 mg of BOC-Leu-Phe-OI-I (1 millimol) and 209.7mg of I-I-Val-OBu I-ICl (l millimol) were dissolved in 2 ml of absolutedimethylformamide or tetrahydrofurane. 0.12 ml of N-ethyl-morpholine (lmillimol) was added and the whole was cooled in an ice bath. Then, theadditives were added and finally asolution that had been cooled to 0 Cof 207 mg of DCC (1 millimol) in 1 ml of absolute dimethylformamide ortetrahydrofurane. The batches were allowed to stand for 5 hours at 0 Cand for about 15 hours at room temperature, then diluted with about 30ml of ethyl acetate. The precipitate that separated was filtered off,the filtrate was shaken with saturated sodium bicarbonate solution, 2Ncitric acid, saturated sodium bicarbonate solution and water, dried withsodium sulfate, and concentrated. The residue was chromatographed inethyl acetate over 3 g of basic aluminum oxide (Woelm, activity degreeI). The eluate (about 40 ml) was concentrated and the residue wasdissolved in about 5 ml of 8 9 N-methanolic hydrochloric acid and heatedfor 24 hours to C in a bomb tube. The methanolic hydrochloric acid wasconcentrated" and the further treatment was effected as described by F.Weygand et al.

The results are indicated in following Table l. T a b l e l Racemizationtests in the dicyclohexyl-carbodiimide method with different additivesEquiv- Solu- D-Phealent Additive tions L-Val 0 none DM F 14.3 0 none THF8. 1 2 penta-chlorophenol DMF 27.0 2 penta-chlorophenol THF 10.0 1N-hydroxy-succinimide DN F 1.0 1.2 N-hydroxy-succinimide DMF 10 2N-hydroxy-succinimide DM F 1.0

N N---.-NN;--- N NN M v- 2. Test for racemization in the activation ofpeptides with dicyclohexyl'carbodilmide and y y' 5 tion. The filtratewas concentrated and the residue was benzotriazole orN-hydroxy-succinimide 378.5 mg of BOC-Leu-Phe-OH (1 millimol) and theadditive were dissolved in 2 ml of absolute DMF or tetrahydrofurane. Thewhole was cooled to 0 C. A solution that had been cooled to 0 C of 207mg of 55 11cc (1 millimol) in 1 ml of absolute DMF or tetrahydrofuranewas added and the mixture was allowed to stand for 2 hours at 0 C. Then209.7 mg of H- Val-OBu HCl (1 millimol) and 0.12 ml of N-ethylmorpholine(l millimol were added. The whole was 60 'again'allowed to stand for 3hours at 0 C and for 15 hours at room temperature. The batch was thenN-hydroxy-succirfirfiide TH F l-hydroxybenzotriazole DMFl-hydroxybenzotriazole DM F l-hydroxybenzotriazole DMFl-hydroxybenzotriazole THF 1-hydroxy-fi-nitro-benzotriazole DMF1-hydroxy-fi-nitro-benzotriazole DMF 5-chlorol-hydroxy-benzotriazole DMF5-chlorol-hydroxy-benzotriazole DMF 6-ch1orol-hydroxy-benzotriazole DMF6-chloro' l-hydroxy-ben z otriazole 12M} 7 5 .6-dichlorolhydroxybenzotriazole DMF DMF -bromol hydroxy-benzotriazole DMF DMF l.01hydroxy-6-trifluoromethylbenzotriazole DMF 1-hydroxy-6-benzotriazolesulfonamide DMF 1-hyd roxy-6-benzotriazolesulfonamide DMFlhydroxy-6-benzotriazolesulfonicacid diethyl amide DMF DMF1hydroxy-G-benzotriazolesulfonicacid methyl amide DMF DMF1-hydroxy-6-benzotriazolecarboxamide DMF DMF1-hydroxy-6-methyl-benzotriazole DMF DMF 1-hydroxy-5methyl-benzotriazoleDMF DMF 1hydroxy-4-methylbenzotriazole DMF DMF 1 hydroxy-S.6-dimethylbenzotriazole DMF l-hydroxy- 5,6-dimethylbenzotriazole DMFl-hydroxy-S-methoxybenzotriazole DMF DMF1-hydr0xy-6-methoxybenzotriazole DMF 1.0 DMFlhydroxy-6-methyl-5benzotriazole-carbonitrile DMF DMF4-chloro-1hydroxy-7-methyl-6- nitro-benzotriazole DMF DMF 11hydroxy-4-methyl-6-nitrobenzotriazole DMF DMF6-chloro-1hydroxy-S-methylbenzotriazole DMF DMF6-chloro-1hydroxy-S-isopropylbenzotriazole DMF DMF4,5,6,7-tetrachloro-l-hydroxybenzotriazole DMF worked up as described inExample I l.

The results are given in following Table 2.

T a b l e 2 Tests for racemization in the activation of peptides withdicyclohexyl-carbodiim ide and l-hydroxy- 38 g of phenyl-alanine weredissolved in 345 ml of dioxane and 115 ml of 2N sodium hydroxidesolution. 38 g of BOCLeu-OSu were added and the whole was stirred forabout 20 hours at room temperature. The l.0 precipitate (excessphenyl-alanine) was filtered off 15 with suction and the filtrate wasconcentrated. The 1.0 residue was distributed between 200 ml of ethylacetate 10 and 220 ml of 2N citric acid. The ethyl acetate phase 1.0 wasonce again shaken with 2N citric acid and water, l0 dried with sodiumsulfate and concentrated. The 1I0 residue was triturated with petroleumether. Yield:

2- 39.3 g; melting point: 108- 1 1 1C. After recrystallization from amixture of ethyl acetate and petroleum g8 ether: yield 33.3 g. Meltingpoint: 1l2 ll5C, [a] j 8.5(c=2, methanol).

4. Preparation of H-Val-OBu' HCl NCarbobenzoxy-L-valine-tert.buty1 esterwas 1.3 hydrogenated catalytically in methanol with a palladi- 1 0 umcatalyst and with the aid of an autotitrator at pH 5 (addition of 1N-methanolic HCl).

H-Val-OBu HCl can be recrystallized from ethyl 1'65 acetate. Meltingpoint: 148 C, [01],, +l9 .22 C (c 2, methanol).

' ll. Synthesis of Z-Val-Val-OCH according to thedicyclohexyl-carbodiimide method with different 1 additives A. One-PotMethod (General prescription) 2 40 2.5 g of Z-Val-OH (l0 millimols 1.7 gof H-Val- OMe HCl (10 millimols 1.28 ml of N-ethylmorpholine( 10millimols and 10 20 millimols of a substituted 1-hydroxy-benzotriazolewere cooled to 0 1 C in 20 ml of absolute tetrahydrofurane, whilestirring. A solution that had been cooled to 0 C of 2.2 g of DCC 1.4 inabsolute tetrahydrofurane was added and the whole was stirred for onehour at 0 C and for 1 hour at room temperature. The precipitate wasfiltered off with sucdissolved in ethyl acetate. The solution was washedwith saturated sodium bicarbonate solution, 2N hydrochloric acid,saturated sodium bicarbonate solution and water, dried with sodiumsulfate, concen trated. The residue was triturated with petroleum ether.The results obtained with di-peptide ester using the differentl-hydroxy-benzotriazoles as additives are given in Table 3.

B. Pre-activation of the carboxyl component (General procedure) 2.5 g ofZ-Val-OH (10 millimols and 10 20 miladded to 10 ml of absolutetetrahydrofurane and combined, at 0 C, with a cold solution of 2.2 g ofDCC in 5 absolute tetrahydrofurane. The whole was allowed to stand for 1hour at 0 C and for 1 hour at room temperature, 1.7 g of H-Val-OMe.l-1Cl(l0 millimols and limols of a substituted lhydroxy-benzotriazole were1.28 ml of N-ethyl-morpholine (10 millimols were added and the mixturewas stirred for 1 hour at room temperature. Working up was effected asdescribed under A. The results obtained with di-peptide ester using thedifferent l-hydroxy-benzotriazoles as additives are given in Table 3.

T a b l e 3 Preparation of Z-Val-Val-OMe Yield No.

Method Additive (C) Equiv. l A 2 l-hydroxy-6-benzotriazole sull'onicacid diethyl amide 90.5 97-100 2 A 2 l-hydroxy-6-benzotriazole sulfonicacid methyl amide 96.1 99-102 3 A 2 l-hydroxy-6-benzotriazole sulfonicacid amide 905 100-102 4 A 2 l-hydroxy-6-trif1uormethylbenzotriazole90.5 94-97 5 A 2 l-hydroxy-6-methoxybenzotriazole 65.9 98-101 6 B 179.0103-104 7 A 2 1-hydroxy-5-methoxy- I benzotriazole 80.0 107-109 8 B1 83.5 98-100 9 A 2 1-hydroxy-5.6-dimethyl benzotriazole 98.8 98-100 10B 1 93.3102-103 11 A 2 l-hydroxy-4-methyl-benzotriazole 82.4 103-105 12B 1 68.6102-103 13 A 2 1-hydroxy-5-methyl-benzotriazo1e 79.6 104-105 14B 1 71.5104-105' 15 A 2 1-hydroxy-6-nitro-benzotriazole 90.6 93-95 16 A2 6-chloro-1-hydroxy-benzotriazole 96.0 93-98 17 A- 25-chl0ro-l-hydroxy-benzotriazole 90.6 97-100 18 A 25,6-dichloro-l-hydroxy benzotriazole 93.3 93-97 19 A 26-bromo-l-hydroxy-benzotriazole 90.5 100-102 20 A 24-chloro-1-hydroxy7-methyl6- nitro-benzotriazole 80.0 96-99 21 A 26-chloro-l-hydroxy-5-methylbenzotriazole 91.0 95-99 22 A 2l-hydroxy-6-methyl-5- benzotriazole-carbonitrile 66.0 103-106l-hydroxybenzotriazole 82.4 92-103 24 A 93.4 99-102 25 B 1 90.7103-107-26 A without addition 70.0 99-107 27 A 2 N-hydroxy succinimide 68.776-82 28 B 72.8 74-85 29 B 1.1 81.0 82-86 111. Synthesis ofBOC-GiN-(Bz)-OBz 1. With unisolated BOC-G1u (OBT)OBz;

To a solution cooled to 0 C of 3.4 g of BOC-Glu- 0B2 (10 millimoles) and1.5 g of l-hydroxybenzotriazole (ll millimoles) in 20 ml of absolutetetrahydrofurane., a solution which. had likewise been cooled to 0 C of2.2 g of DCC in absolute tetrahydrofurane was added. The whole wasallowed to stand for 1 hour at 0 C and for 1 hour at room temperature1.1 ml of benzylamine was added and the mixture was allowed to stand for1 hour at room temperature. The precipitate was then filtered off withsuction and the filtrate was concentrated. The residue was dissolved inethfl'acetate and the solution was shaken successively with saturatedsodium bicarbonate solution, 2N citric acid, saturated sodiumbicarbonate solution and water, dried with sodium sulfate andconcentrated. The residue was triturated with petroleum ether. Yield:3.6 g (84.4 percent); melting point: 87 88 C. For further purification,the substance was dissolved in ethyl acetate and chromatographed onabout 20 g of basic aluminum oxide (Woelm, activity degree 1). Theeluate was concentrated and the residue was triturated .with petroleumether. Yield: 2.9 g (68.1 percent). The substance was found to bechromatographically'pure. Melting point: 92 94 C; [01],, 19.9 (C 2,methanol).

2. With unisolated BOC-Glu(OSu)-OBz (with 2 equivalents ofN-hydroxy-succinimide) The batch was prepared as described under 111 1.Instead of 1-hydroxy-benzotriazole, 2.3 g of N-hydroxysuccinimide (20millimoles) were added. The residue was not crystallizable withpetroleum ether. After purification over a basic aluminum hydroxide 1.75g (41.4 percent) crystallized. Melting point: 75 77 C. The substance waschromatographically notpure.

3. With unisolated BOC-Glu(OSu)OBz (with 1 equivalent ofN-hydroxy-succinimide) The batch was prepared as described under 111 1.Instead of l-hydroxy-benzotriazole, 1.15 g of N-hydroxysuccinimide (l0millimoles) wereadded. The residue was not crystallizable with petroleumether. After purification over a basic aluminum oxide column, 2.2 g(51.5 percent) crystallized; melting point: 88 90 C. 1n thin-layerchromatography, the substance was almost pure.

1V. Peptide Syntheses with Z-Phe-OBT 1. Z-Phe-OBT A solution cooled to 0C of 22 g of DCC was added to a solution, which had likewise been cooledto 0 C, of 30g of Z-Phe-OH (0.1 mol) and 15 g of l-hydroxybenzotriazole(0.1 1 mol) in 300 ml of absolute tetrahydrofurane. The whole wasallowed to stand for 1 hour at 0 C and for 1 hour at room temperature.The precipitate was filtered off with suction and the filtrate wasconcentrated. The residue was recrystallized from isopropanol. Yield:28.9 g (69.5 percent); melting point: l20- 122C.

2. Z-Phe-Phe-OCH 1.2 g of l-l-Phe-OCH HCl (5 millimols) were suspendedin 20 ml of absolute tetrahydrofurane. The suspension was cooled to 0 C,0.7 ml of N-ethylmorpholine (5 millimols) was added and the whole wasstirred for 5 minutes. Then, 2.1 g ofZ-Phe-OBT were 1 added and themixture was stirred overnight. On the acetate. Yield: 1.7 g (82.5percent); melting point:

111 113 C; [01],, =8.9 (c 2, in dimethylformamidejl H 4. Kinetic teststo the synthesis of Z-Phe-Val-OCH 115.2 mg of N-ethyl-morpholine (lmillimol) were dissolved in ml of absolute tetrahydrofurane that hadbeen cooled to 0 C. The solution was combined with 184 mg of l-l-VaLOCHHCl (1.1 millimol) and then,

with stirring, with 458 mg of Z-Phe-OBT 1.1 millimol) or 436 mg ofZ-Phe-OSu (1.1 millimol). The whole was then stirred at 0 C. Thereaction was stopped at the desired time by the addition of ml of O.l-N

hydrochloric acid. The free hydrochloric acid was then determinedpotentiometrically with 0.1-N sodiuml hydroxide solution with the aid ofa glass electrodel The consumed quantity of 0.1-N-sodium hydroxidelsolution is proportional to the yield of peptide. The accompanyingdrawing shows the results of these kinetic tests.

V. Synthesis of a protected deca-peptide having the sequence ofantamanide (Z-Phe-Phe-Pro-Pro-Phe- Phe-Val-Pro-Pro-Ala-OBu) 1.Z-Pro-Pro-OH (MW 346) 370 g of Z-Pro-ONP (1 mol) and 127 g (1.1 mol) ofPro were boiled in the presence of 140 ml (1 mol) of triethylamine for 4hours in ethanol. The solvent was removed by distillation under reducedpressure, the residue was dissolved in 1.5 liter of water and extractedtwice with each time 250 ml of ether. The aqueous phase was acidifiedwith semi-concentrated HCl to pH 2, whereupon a crystalline precipitatewas obtained which was recrystallized from a small amount of ethanol.Yield: 256 g (74 percent); melting point 188 189 C; [a],, =10l(c =2, in50 DMF).

2. H-Pro-Pro-Ol-l (MW 212) 60 g (0.173 mol) of Z-Pro-Pro-OH werehydrogenated in 500 ml of 80 percent methanol in the presence ofpalladium catalyst. After separation of the catalyst by filtration, thesolvent was removed by distillation under reduced pressure, the residuewas triturated with acetone, filtered off and washed with ether.

Yield: 35.2 g (96 of the theory); [01],, 2, in water).

3. Z-Phe-Phe-OH (MW 446) 66 g of Phe (0.4 mol) were dissolved in amixture of 800 ml of dioxane and 200 ml of 2N-NaOl-l.79.2 g (0.2 mol) ofZ-Phe-OSu were added and the whole was stirred overnight at roomtemperature. The Phe that had precipitated was filtered off. Thefiltrate was evaporated to dryness under reduced pressure, the residuewas dissolved in 400 ml of ethyl acetate, shaken with 2N-HC1 and water,the ethyl acetate phase was dried over sodium sulfate and and thesolvent was removed by distillation. The residue was recrystallized froma mixture of ethanol and water (3 1). Yield: 69.0

g (77.4 percent of the theory); melting point: 158

recrystallized from ethanol. Yield: 42.555 cent); meltingpoint: 148 151C; [a] 30.9 (c 1, in DMA). 37.8 g millimols) of this hydroxysuccinimideester and 15.4 g (73.5 millimols) of l-l-Pro- Pro-OH were stirred in mlof DMF. After 45 minutes, the total amount of H-Pro-Pro-OH had passedinto solution. 8.1 g 70 millimols) of N-ethylmorpholine were added andthe whole was stirred overnight at room temperature. After removal ofthe solvent by distillation under reduced pressure, the residue wasdissolved in 200 ml of ethyl acetate, shaken with lN-HCl and a smallamount of water, dried over sodium sulfate and the ethyl acetate wasremoved by distillation. The residue was dissolved in a small amount ofacetone. Upon addition of 8.5 ml of cyclohexylamine and 300 ml of ethera precipitate separated which, after standing for several hours at 0 C,was filtered off, dried and recrystallized from isopropanol. Yield: 29.0g (64.8 percent); melting point: 194 196 C; [01],, l01(c= 0.3 inmethanol). C., H N O (739.8)

Calc.: C 68.2 H 7.08 N 9.47

Found: 068.2 H 7.2 N 9.6

"'F'di'siftiiifiiiii acid, the compound was dissolved in ethyl acetate,the solution was shaken twice with each time 50 ml of lN-HCl washed witha small amount of water, dried over sodium sulfate and the ethyl acetatewas removed by distillation under reduced pressure. The yield was almostquantitative.

5. Synthesis of l'l-Pro-Pro-Ala-OBu l-lCl.

a. According to the dicyclohexyl-carbodiimide method with the additionof 2 equivalents of 1- hydroxy-benzotriazole A cold solution of 2.2 g ofDCC in absolute DMF was added at 0 C to a solution of 3.46 g ofZ-Pro-Pro-Ol-l (10 millimols), 1.82 g of l-l-Ala-OBu HCl (10 millimols),2.7 g of l-hydroxy-benzotriazole (20 millimoles) and 1.28 ml ofN-ethyl-morpholine (10 millimols) in 30 m1 of absolute DMF. The wholewas allowed to stand for 2 hours at 0 C and for 1 hour at roomtemperature. The precipitate was filtered off, the solution wasconcentrated, and the residue was distributed between ethyl acetate andwater. The ethyl acetate phase was washed successively with saturatedsodium bicarbonate solution, 2N citric acid, saturated sodiumbicarbonate solution and water, dried with sodium sulfate andconcentrated. The residue was chromatographed over about 20 g of basicaluminum oxide (Woelm, activity degree I) in ethyl acetate. The eluatewas concentrated. The yield of oily Z-Pro-Pro- Ala-OBu was 4.0 g (84.5percent).

The oily Z-Pro-Pro-Ala-OBu was hydrogenated catalytically in methanolwith the addition of a palladium catalyst. A pH of 5 was maintained withthe aid of as" aii tdtit'rat'of by the addition of'W-Ttlfiidfi?hydrochloric acid. When the reaction was complete (no further uptake ofmethanolic hydrochloric acid), the catalyst was removed by filtration.The filtrate was concentrated and triturated with ether. Yield: 2.75 g(73.2 referred to Z-Pro-Pro-Ol-l). This substance waschromatographically uniform. Melting point: 147 151 C; [a],,= 152 (0 =2,in methanol).

b. According to the dicyclohexyl-carbodiimide method with the additionof 2 equivalents of N- hydroxy-succinimide..

The batch was prepared as described under V 5a. Instead ofl-hydroxy-benzotriazole, 2.3 g of N-hydroxysuccinimide (20 millimoles)were added. Yield of oily Z-Pro-Pro-Ala-OBu': 3.1 g (65.5 percent).

After catalytic hydrogenation, 1.8 g of l-l-Pro-Pro- Ala-OBu HCl couldbe isolated (47.8 referred to Z-Pro-Pro-Ol-l). Melting point: 147 151 C.A ninhydrin negative substance could be detected in traces by thin-layerchromatography.

c. With not isolated Z-Pro-Pro-OBT A solution of 2.2 g of DCC, dissolvedin cold DMF, was added at C to a solution of 3.46 g of Z-Prop-Pro (10millimoles) and 1.5 g of l-hydroxy-benzotriazole (11 millimoles) in 30ml of absolute DMF. The whole 15 was allowed to stand for 1 hour at 0 Cand for 1 hours at room temperature. Then 1.82 g of H-Ala-OBu' HCl' (10millimoles) and 1.28 ml of N-ethyl-morpholine (l0 millimoles) wereadded. After a further hour at room temperature, working up was effectedas described 011) were obtained. Melting point: 145 150 C. The

substance was found to be chromatographically uniform.

6. Z-Val-Pro-Pro-Ala-OBu A' Eoldbluiini 654.5513? DCC in 5155mm;-

tetrahydrofurane was added at 0 C to a solution of 4.6 g of Z-Val-Oh(18.3 millimols) and 2.75 g of l-hydroxy-benzotriazole (20.4 millimols)in ml of absolute tetrahydrofurane. The whole was allowed to stand for 1hour at 0 C and for 1 hour at room temperature. Then, 5.8 g ofl-l-Pro-Pro-Ala-OBu HCl (15.5 millimols) and 1.98 ml ofN-ethyl-morpholine (15.5 millimols) were added with stirring. The wholewas stirred for 1 hour at room temperature, the precipitate was filteredoff, the filtrate was concentrated, and working up was effectedasdescribed under V 5a.

Yield: 8.4 g of oil (95 percent).

7. Z-Phe-Phe-Val-Pro-Pro-Ala-OBu 8.4 g of Z-Val-Pro-Pro-Ala-OBu (14.67millimols) 45 complete, the catalyst was removed by filtration, theeluate was concentrated, and the residue was triturated with ether. Theether was decanted off and the oil was dried in a high vacuum. Anamorphous mass of 6.8 g (14.3 millimols) of H-Val-Pro-Pro-Ala-OBu HCl(97.6 percent) was formed which was dissolvedi together with 6.45 g ofZ-Phe-Phe-OH (14.4 millimols), 3.85 g'of 1 5yarbrfiafizbifiazalwfifiilimols) and 1.83 ml of N-ethyl-morpholine (14.3 millimols) in 50 ml ofabsolute DMF. A cold solution ofi 3.15 g of DCC in absolute DMF wasadded at 0 C and the whole was stirred for 2 hours at 0 C and for 1 hourat room temperature. The precipitate was filtered off with suction andthe filtrate was concentrated. The

residue was dissolved in ethyl acetate and extracted by her...

Yield: 111E188 percent, refer rEZiTGZ VaI Pro-Prci' Ala-OBu' initiallyused). For purification, the product was chromatographed intetrahydrofurane over basic aluminum oxide (Woelm, activity degree 1).

Yield: 9.05 g (71.1 percent, referred to Z-Val-Pro- Pro-Ala-OBu). Themelting point was unsharp C). [01],, =126 (c 2, in methanol).

The substance was chromatographically uniform.

When, instead of l-hydroxy-benzotriazole, 2 equivalents ofN-hydroxy-succinimide were added 78 of crudeZ-Phe-Phe-Val-Pro-Pro-Ala-OBu were isolated. From this crude substance,only about 75 percent could be recovered after purification overaluminum oxide. [01] =126 (c 2, in methanol).

8. H-Phe-Phe-Val-Pro Pro-Ala-OBu' Palladium catalyst was added to asolution of 8.9 g of Z-Phe-Phe-Pro-Pro-Val-Pro-Pro-Ala-OBu in methanoland hydrogenation was effected as usual while maintaining the pH at 5 byadding methanolic hydrochloric acid with the aid of an autotitrator.When the hydrogenation was complete, the catalyst was removed byfiltration and the filtrate was concentrated under reduced pressure. Theresidue was dissolved in water and insoluble matter was filtered off.The filtrate was adjusted to pH 9 by means of sodium bicarbonatesolution and the oil that precipitate was extracted thrice with ethylacetate. The combined ethyl acetate phases were dried with sodiumsulfate and concentrated. 7.3 g of an amorphous substance remainedbehind (97.4 percent). The substance was found to be chromatographicallyuniform.

9. Z-Phe-Phe-Pro-Pro-Phe-Phe-Val-Pro-Pro-Ala- OBu' a. According to thedicyclohexyl-carbodiimide method with addition of 2 equivalents ofl-hydrox-v y-benzotriazole I p 8.1 g, of Z-Phe-Phe-Pro-Pro-Ol-l CHA (11millimols) were distributed between ethyl acetate and 2N hydrochloricacid. The-ethyl acetate phase was washed with water, dried over sodiumsulfate and concentrated. The residue was dissolved in 40 ml of DMF andcombined with 7.3 g of H-Phe-Phe-Va1-Pro-Pro-A1a- OBu (10 millimols) and2.7 g of l-hydroxybenzotriazole (20 millimols). The whole was cooled to--10 C and a solution of 2.2 g of DCC, dissolved in cold DMF, was added.The mixture was allowed to stand for 4 hours at a very low temperatureand then overnight at room temperature. On the next day, it wasconcentrated and the residue was dissolved in ethyl acetate andextracted as described under V 5a. The residue was dissolved intetrahydrofurane and chromatographed over a basic aluminum oxide column.The eluate was concentrated and dried in a high vacuum. An amorphoussubstance remained behind which was found to be thin-layerchromatographically uniform.

2.1 g of Z-Gly-NH and 2.4 g of 4,4'-dimethoxy benzhydrol were dissolvedin ml of glacial acetic acid. One drop of concentrated H2804 was addedand the whole was allowed to stand overnight. On the next day it wasdiluted with 40 ml of water and cooled. The

crystal m a g rna fiiat precipitated was filteredbfi witli acid andhydrogenated catalytically with a palladium catalyst. The catalyst wasthen removed by filtration with suction and the filtrate wasconcentrated. The residue was dissolved in methanol and titrated withmethanolic hydrochloric acid against thymol blue. Concentration wasrepeated and the residue was triturated with ether. Yield: 33.9 g (98percent). After recrystallization from a mixture of methanol and ether:31.7 g (91.6 percent). Melting point: 202- 204 C.

b. From chloroacetic acid-4,4'-dimethoxy-benzhydrylamide: 18.7 g ofchloroacetic acid amide and 48 g of 4,4- dimethoxy-benzhydrol werereacted in 200 ml of glacial acetic acid and 0.5 ml of concentrated H SOas described for the preparation of Z-Gly-NM-Mbh. Yield: 45.75 g (72%);melting point: l126 C.

220 ml of condensed ammonia were added to a solution of 88.5 g ofchloroacetic acid-4,4'-dimethoxy-' benzhydryl amide in 880 ml ofmethanol and the whole was heated in an autoclave for 24 hours to 60 70C. It was then concentrated and the residue was distributed betweenethyl acetate and a sodium bicar-' bonate solution. The ethyl acetatephase was washed with water, dried with sodium sulfate and concentrated.The residue was dissolved in methanol and combined with methanolichydrochloric acid until a pH of 6 was reached. Concentration wascontinued and the residue was triturated with ether. Melting point: 173176 C.

For purification, the product was dissolved in water and treated withanimal charcoal. The clear solution in water was concentrated and theresidue was recrystallized from a mixture of methanol and ether. Yield:59.3 g (63.6 percent). Melting point: 197 199 C.

6.1 g of Z-Lys-(BOC)-OH DCl-lA (11 millimols) were distributed betweenethyl acetate and 20 ml of 2N Nl-l-Mbh HCl (10 millimols) and 1.3 ml ofN-ethylmorpholine (10 millimols) were added. The mixture was stirred for1 hour at room temperature. The precipitate was filtered off withsuction, the filtrate was concentrated. The residue was dissolved inwarm ethyl acetate and a sodium bicarbonate solution. The ethyl acetatesolution was shaken with 2N citric acid, saturated sodium bicarbonatesolution and water, dried with sodium sulfate and concentrated.Tlieresidue was recrystallized from a mixture of ethyl acetate andpetroleum ether.

Yield: 6.0 g (90.7 percent); melting point 123 125 C; [a],,=+8.9 (c 2,in methanol).

3. H-Lys-(BOC)Gly-NH-Mbh HCl 9.6 g of Z-Lys(BOC)-Gly-NH-Mbh weresuspended in methanol. A palladium catalyst was added and catalytichydrogenation was effected while adding dropwise lN-methanolichydrochloric acid (autotitrator, pH 5). The catalyst was removed byfiltration with suction and the solution was concentrated. The residuewas triturated with ether.

Yield: 7.3 g (89 percent). After recrystallization from a mixture ofmethanol and ether, the melting point was at 158 160 C. [04] +l7.55 (c2, in methanol).

4. Z-Pro-Lys(BOC)-Gly-NH-Mbh 2.6 ml (20 millimols) of N-ethyl-morpholinewere added at0 C and then, while stirring, a cold solution of 4.4 g ofDCC in DMF was added to a suspension of 5 g of Z-Pro-Ol-l (20millimols), 11.4 g of H-Lys-(BOC) Gly-NH-Mbh HCl (20 millimols) and 5.4g of lhydroxy-benzotriazole (40 millimols) in 60 ml of DMF. The wholewas stirred for 1 hour at 0 C and for 1 hour at room temperature. Theprecipitate was filtered off with suction and the filtrate wasconcentrated and worked up as describedunder V 2.

Yield: 14.1 g (93 percent); melting point: 180 183 C. Recrystallizablefrom a mixture of methanol and water. [a] =27.4 (c 2, in DMF).

5. H-Pro-Lys(BOC)-Gly-NH-Mbh -HC1 18.5 g of Z-Pro-Lys(BoCyGly-Nh-Mbhwere hydrogenated catalytically in methanol as described under VI 3.

lonwards (amorphous). 6. Z-AsN(Mbh)-OH l 0.5 ml of concentrated H SO wasadded at room temperature to a solution of 27 g of Z-Asn-OH and 24 g of4,4-dimethoxy-benzhydrol in 250 ml of glacial acetic acid. The whole wasallowed to stand overnight at room temperature and then poured into 750ml of water. The crystal magma that precipitated was filtered off withsuction and dissolved hot in ethyl acetate. The ethyl acetate solutionwas shaken with water, dried with sodium sulfate and evaporated underreduced pressure. The residue was tirturated with ether and filtered offwith suction. Yield: 47.5 g (96 percent); melting point: 176 180 C;[01],, +2.4 (c 2, in dimethylformamide).

49.2 g of Z-AsN(Mbh)-OH were suspended in 400 ml of glacial acetic acidand hydrogenated catalytically in the usual manner. After completedhydrogenation, the catalyst was removed by filtration and the filtratewas concentrated under reduced pressure. The residue was triturated withsodium acetate solution, filtered with suction, washed thoroughly withwater and dried over P 0 Yield: 33.6 g (94 percent); melting point:

Yield: 15.2 g (100 percent) sintering from 100 C solution were addedportionwise, while maintaining the pH at 8. The mixture was then dilutedwith 930 ml of water, acidified with citric acid (pH 3) and the aqueousphase was extracted twice by shaking with ethyl acetate. The ethylacetate was dried with sodium sulfate and combined with cyclohexylamineuntil a basic reaction could be observed. A yellow crystal magmaprecipitated which was filtered off with suction and washed with ethylacetate. Yield: 47.5 g (85 percent); melting point: 182 184 C; [01],,=29.8 (c 1, in dimethylformamide).

NPS-AsN-Mbh)-OSu 18.6 g of NPS-AsN(Mbh)-Ol-l Cl-lA (30.5 millimols) weredistributed between ethyl acetate and about 80 ml of 2N citric acid atC. The ethyl acetate phase was washed with 2N citric acid and water,dried with sodium sulfate and concentrated. The residue was dissolved in100 ml of tetrahydrofurane, combined with 3.45 g ofN-hydroxy-succinimide (30 millimols), cooled to 0 C and mixed with asolution of 6.2 g of DCC in cold tetrahydrofurane. The whole was stirredfor 1 hour at 0 C and for 1 hour at room temperature, and was thencombined with a small amount of dimethylformamide. The dicyclohexyl-ureathat precipitated was filtered with suction and the filtrate wasconcentrated. The residue was boiled up with isopropanol. Yield: 14.3 g(77 percent); melting point:

2.5 g ofNPS-AsN(Mbh)-OSu were added, while stirring, to a solution of2.25 g of H-Cys(Mmb)-OH (8.2 millimols) in 20 ml of dioxane and 4. 1. mlof 2N sodium hydroxide solution and the whole was allowed to stand"phase was washed once with 2N citric acid and once.

with water and dried with sodium sulfate. The salt was precipitated withcyclohexylamine.

Yield: 2.45 g (69.5 percent); melting point: 198 202C.

8. NPS-AsN(Mbh)-Cys(Mmb)-Pro-Lys(BOC)-Gly- 4.3 g ofNPS-AsN(Mbh)-Cys(Mmb)-Ol-l-CHA (5' millimols) were distributed whilecooling with ice between ethyl acetate and 2N citric acid. The ethylacetate phase was washed with 2N citric acid and water, dried withsodium sulfate and concentrated. The residue was dissolved in ml of DMF.The solution was combined with 3.15 g of l-l-Pro-Lys(BOC)-Gly-Nl-l-Mbhl-lCl (5 millimols), 0.64 ml of N-ethylmorpholine (5 millimols)and 0.7 g of l-hydroxybenzotriazole (5 millimoles), cooled to 0 C andcom-. bmed, with stirring, with 1.1 g of DCC dissolved in a small amountof cold DMF. The whole was stirred for 1 hour at 0 C and for 1 hour atroom temperature. The precipitate was filtered off with suction and thefiltrate was concentrated. The residue was dissolved in ethyl acetateand extracted by shaking as described under V1 2. The residue waschromatographed over neutral aluminum oxide (Woelm, activity degree I)in tetrahydrofurane.

Yield: 4.95 g (77 percent); melting point: 160

164C; [01],, -12.45 (c 2, in dimethylformamide).

Vll. Synthesis of asparaginyland glutaminyl-peptides 1. Z-AsN-Leu-OCH1.28 ml of N- ethyl-morpholine (10 millimoles) and, finally, a coldsolution of 2.1 g of DCC in a small amount of DMF were added at 0 C to asolution of 2.7 g of Z-AsN-OH (10 millimols), 1.7 g of l-l-Leu- OCH-l-ICI (10 millimols) and 2.7 g of l-hydroxybenzotriazole in 30 ml ofDMF. The whole was stirred for 1 hour at 0 C and for 1 hour at roomtemperature. The precipitate was filtered off with suction and thefiltrate was combined with water. The precipitate that had separated wasfiltered off with suction, triturated with sodium bicarbonate solution,again filtered off withsuction' and washed with water and dried overphosphorus pentoxide.

Yield: 3.35 g percent); melting point: 176 C; [a] =26.3(c=2, inmethanol).

2. z-GiN-Ala-OBu 1.28 ml of N-ethyl-morpholine (10 millimols) and,finally, a cold solution of 2.1 g of DCC in a small amount of DMF wereadded at 0 C to a solution of-2.8 g of Z-GlN-Ol-l (10.millimo1s), 1.8 gof H-Ala-OBu HCl (10 millimols) and 1.35 g of 1l-lydroxy-1,2,3-benzotriazole (10 millimols) in DMF. Further operations were asdescribed under V11 1.

v Yield: 3.0 g (73.7 melting point: 158 161 C; [a],,=36.0 (c=2, inmethanol).

When 2 equivalents of l-hydroxy-l,2,3- benzotriazole were added, theyield was 2.9 g (71.2 percent); melting point: 158 161 C.

Vlll. Z-Ser-Gly-ONB 1.3 ml of ethyl-morpholine and, finally, a coldsolution of 2.2g of DCC in DMF were added at 0 C to a solution'of 2.4 gof Z-Ser-Ol-l 10 millimols), 2.7 g of 1- hydroxy-benzotriazole (20millimols) and 2.9 g of H- Gly-ONB HBr in 30 ml of DMF. The whole wasstirred for 1 hour at 0 C and for 1 hour at room temperature and thenworked up as described under 11 1.

Yield: 4.2 g (97.5 percent); melting point: 121 123 C; [01],, =-8.2 (c2, in glacial acetic acid).

1X. Corticotropin-( 1-23) -tricosa-peptide-amide 8.25 g ofBOC-Ser-Tyr-Ser-Met-Glu(OBu')-l-lis- PheAgr-Trp-Gyl-Ol-l (5.5millimols), prepared according to Chem. Ber. 96, 1,080, (1963), and 12.5g (5 mil-- limols) of l-1-Lys(BOC)-Pro-Va1-G1y-Lys-(Boc)-Lys(Boc)-Arg-Arg-Pro-Val-Lys(Boc)-Val-Tyr-Nl-1 tritosylate, whichhad beenobtained from the acetate prepared according to Chem. Ber. 97, 1197(1964) by adding the calculated amount of toluenesulfonic acid in water,evaporation of the solvent under reduced pres-' sure and dissolving andreprecipitating the residue from a mixture of pyridine and ether, weredissolved in 150 ml of DMF. After the addition of 2.7 g ofl-hydroxybenzotriazole (20 millimoles), one third of the solution of 6.5g (30 millimols) of DCC in 20 ml of DMF was added at room temperature.After 1 hour, a further third and after another hour the last third ofthe DCC solution were added. After 2 hours the crude reaction productwas precipitated with ether. Yield: 19.9 g. The protective groups weresplit off in known manner by treatment for 1 hour with percenttrifluoracetic acid which contained a small amount of thio-glycolicacid. The crude tricosapeptide was precipitated by means of 'stirred for2 hours at room temperature. The solventether and washed with ether.Yield: 19.1 g. For purifi cation, the product was chromatographed inknown manner on carboxy-methyl cellulose.

X. Z-Leu-Ala-Leu-Glu(OBu')-Gly-Pro-Pro-GIN- Lys(BOC)-Arg-Ol-l (Sequence54 63 of pigs pro-insulin (The preparation of the starting peptides isdescribed hereinafter) 11.5 g (18 millimols) ofZ-Leu-Ala-Leu-Glu-(OBu')- OH and 2.43 g (18 millimols) ofl-hydroxybenzotriazole in 80 ml of DMF were combined at -l0 C whilestirring, with 3.7 g (18 millimols) of DCC in 25 ml of DMF. The wholewas stirred for 2 hours at room temperature freed from urea byfiltration and combined with the solution of 13.3 g millimols) of H-Gly-Pro-Pro-GlN-Lys(Boc)-Arg-OH, 115 ml of Ch COOH, H 0 and 1.92 mil (15millimols) of N- ethyl-morpholine in 80 ml of DMF. The whole was wasremoved by distillation under reduced pressure and the residue wastriturated with ethyl acetate. Yield of crude product: 17.1 g. Thecompound was boiled thrice with ethyl acetate. The dried substance wasthen triturated thoroughly with water. Yield: 15.5 g (73.5 percent).Thin-layer chromatography: impurities in traces only. [04],, 80.0 C (c2, in methanol).

Amino-acid analysis:

Cale: G lu FY02 GlyI AlflLeu} Lys Arg eFe p z. -eP (Yo-$ Alan aste.

osz

NH Calc.: 1 equivalent Found: 1.09 equivalent Preparation of thestarting products for Example X:

a.Z-Lys(Boc)-Arg(NO )-OCH (596) 57 g (0.2 mol) of H-Arg(NO )-OCH HClwere stirred overnight with 95 g (0.2 mol) of Z-Lys-(BOC)- OSu in 500 mlof DMF. After removal of the solvent by distillation under reducedpressure, a resin remained behind which was dissolved in ethyl acetateand extracted at 0 C with 2N citric acid, bicarbonate and water. Thesolution was dried over Na SO and the SOl-l vent was removed bydistillation under reduced pres-' sure. The residue formed a jelly whichwas trituratedi with ether and dried at the air. Yield: 106 g (89 per-?cent). TC: pure. For analysis, a sample of the product wasrecrystallized from ethyl acetate. Melting point; 130 132 C; [a],, =12.5(c l, in methanol). C I-1 N 0 (595.6); i Calc.C=52.4l-l=6.94N= 16.5FoundC=52.4H=7.l N=16.7. b.Z-Lys(BOC)-Arg(NO )-OH (581.6) 89 g (0.15mol) of the ester prepared according to (a) were dissolved in a mixtureof 240 ml of water and 500 ml of dioxane. The solution was combined with180 ml of lN-NaOH, stirred for 45 minutes at room temperature andrendered neutral by means of 30 ml of lN-HCl. The dioirane was removedby distillation under reduced pressure. The water phase was extractedtwice with ethyl acetate and acidified'with precaution of 0 C with 150ml of lN-l-lCl. Thereupon an oil separated which was dissolved in ethylacetate. After washing of the solution with water, the solvent wasremoved by distillation with the addition of benzene. The resinousresidue solidified upon trituration with petroleum ether. Yield: 79 g(91 percent). TC: almost pure, a trace of ester. A sample of the productwas boiled with ethyl acetate; TC: pure. Melting point: 168

Calc.: C=51.6 H=6.76 N= 16.86

Found:C=51.6 H=6.8 N= 16.7.

c. H-Lys(Boc)-ArgOl-i 2 CH COOH H 0 75.5 g (0.13 mol) of the di-peptideobtained according to (b) were dissolved in 800 ml of a mixture ofacetic acid, methanol and water (4:422) and hydrogenated catalyticallyon Pd. After removal of the catalyst by filtration, the filtrate wasevaporated under reduced pressure. The residue was triturated with etherand dried over P 0 and KOH. The content of acetic acid was found todepend on the drying conditions. Yield: g (almost quantitative).

Acetic acid: Calc. for 2 mols: 22.2 Found: 21.8 Water: Calc. for 1 mol:3.34 Found: 3.7.

70' (0.13 reiterate di-pe t idobtained according to (c) were stirred for24 hours at room temperature in 600 ml of DMF with the addition of 16.5ml (0.13 mol) of N-ethyl-morpholine with 60 g (0.15 mol) of Z-GiN- ONp.The solvent was removed by distillation under reduced pressure. Theresidue was boiled with ethyl acetate and the insoluble peptide wasfiltered off while hot. For further purification it was dissolved in 150ml of methanol and the solution was combined with 750 ml of 1N-NHwhereupon, the tri-peptide crystallized. Yield: 66.5 g (82 percent) TC:pure.

Arg-Ol-l (858) TheZ tri pEpTiTE ("611 m onfiieaairszzaiaiagta (d) washydrogenated catalytically in 350 ml of DMF with the addition of a largeamount of Pd(BaSO The separation of the Z-group was completed after 2hours.

Then, 62 g (0.115 mol) of Z-Pro-Pro-OSu in ml of DMF was added and thewhole was stirred for 12 hours at room temperature. After removal of thesolvent by distillation under reduced pressure, the residue was boiledin ethyl acetate, triturated with fresh ethyl acetate and washed withether. Yield: 79.9 g. TC: the penta-peptide still contains about 10percent of Pyr- Lys(Boc)-Arg-OH which had been formed from H-GiN-Lys(Boc)-Agr-OH and which cannot be separated in this stage. In thefollowing reactions, however, it did not enter into reaction and couldbe easily split off in a later stage.

Z-Pro-Pro-OSu was prepared as follows:

69.2 g (0.2 mol) of Z-Pro-Pro-OH were suspended in 500 m1 ofCH Cl 27.6 g(0.24 mol) of HOSu were added, the whole was stirred for 10 minutes,cooled to -5 C and combined with 41.2 g (0.2 mol) of DCC in ml of Cl-lcl After stirring for 30 minutes at 0 C and for 2 hours at roomtemperature, the urea was filtered off and the filtrate was evaporatedto dryness palladium catalyst. After removal of the solvent bydistillation under reduced pressure, the residue was triturated withether. Yield: 58.2 g (97.5 percent). TC: obtained as impurity only thePyr-Lys(Boc)-Arg-OH formed under (e).

g. Z-Gly-Pro-Pro-GiN-Lys(Boc)-Agr-OH (916) 36.2 g (50 millimols) ofpenta-peptide were stirred for 24 hours at room temperature in 200 ml ofDMF with 34.8 g (90 millimols) of Z-Gly-OTCP. The solvent was removed bydistillation under reduced pressure. The residue was dissolved in 100 mlof methanol and the reaction product was precipitated by means of 1liter of ethyl acetate. For purification, the product was extractedseveral times with warm water. The aqueous extract was filtered andevaporated under reduced pressure. The residue was again dissolved andreprecipitated from a mixture of methanol and ethyl acetate. Yield: 37.1g (81 percent). TC: in addition to Pyr-Lys(Boc)-Arg-O1-l no, furtherimpurity.

h. l-l-Gly-Pro-Pro-GiN-Lys(Boc)-Arg-OH CH COOl-l H (890) The Z-compoundobtained according to (f) was hydrogenated catalytically in 300 ml of 90percent acetic acid with the aid of palladium catalyst. After removal ofthe catalyst by filtration, evaporation of the filtrate under reducedpressure and trituration of the residue with ether and drying over P Oand K011, the yield was 35 g (97 percent). The content of acetic acidwas not stoichiometric and varied according to the drying conditions.

i. Z-Ala-Leu-Glu(OBu )-OCH 25.4 g of H-Glu(OBu )-OCH HCl, 12.8 ml of N-ethyl-morpholine (0.1 mol) and, finally, a cold solution of 22 g of DCCin a small amount of tetrahydrofurane were added to a solution which hadbeen cooled to 0 C' of 26.5 g of Z-Leu-OH (0.1 mol) and 27 g ofl-hydroxy-benzotriazole (0.2 mol) in 200 ml of tetrahydrofurane. Thewhole was stirred for 1 hour at 0 C and for 1 hour at room temperature.The precipitate was filtered off with suction and the filtrate wasworked up as described under 11 1. The residue was chromatographed inethyl acetate on basic aluminum oxide (Woelm, activity degree 1). Theeluate was concentrated under reduced pressure. Yield: 44.85 g of oil(96.6 percent). The oil was hydrogenated catalytically in methanol asdescribed under V 8. The resulting dipeptide ester hydrochloride wasoily. Yield: 34.8 g (95 millimols). This oil was dissolved with 21.2 gof Z-Ala- OH (95 millimols) and 26.6 g of l-hydroxybenzotriazole (190millimols) in 200 ml of DMF. 12.2 ml of N-ethyl-morpholine (95millimols) and a cold solution of 20.9 g of DCC in DMF were added at 0C. The whole was allowed to stand for 1 hour at 0 C and for 1 hour atroom temperature. The precipitate was filtered off with suction and thefiltrate was worked up as described under 11 l. The residue waschromatographed in tetrahydrofurane over a basic aluminum oxide column.The eluate was concentrated and tritu rated with petroleum ether. Yield:40 g (74.7 percent, referred to Z-Leu-Ol-l). Melting point: 96 98 C. [a]=-54.l (c=2, in methanol).

15.85 g of Z-Ala-Leu-Glu(OBu)-OCH (28.65 millimols) were added to amixture of 100 ml of dioxane and 16 ml of water. 29 ml of lN-sodiumhydroxide solution were added slowly dropwise, within 2 3 hoursneutralized with 2N citric acid and concentrated. The residue wasdistributed between 2N citric acid and ethyl acetate. The ethyl acetatesolution was washed once with water, dried with sodium sulfate andconcentrated. The residue was crystallized from a mixture of ethylacetate and petroleum ether. Yield: 14.05 g (91 percent). Melting point:142 145 C. After recrystallization from a mixture of ethyl acetate andpetroleum ether: melting point: 145 148 C; [01],, 45 (0 2, in methanol).

1. Z-Leu-Ala-Leu-Glu(OBu')-OH l l g of Z-Ala-Leu-Glu(OBu)-Oh weredissolved in a mixture of 50 ml of methanol and 50 ml of glacial aceticacid and hydrogenated catalytically after addition of palladiumcatalyst. The catalyst was filtered off with suction when thehydrogenation was complete, the filtrate was concentrated and theresidue was triturated with ether. Yield: 7.95 g melting point: 214 215C.

The product of the hydrogenation was suspended in 50 ml of DMF. Thissuspension was combined with 7.25 g of Z-Leu-OSu (20% excess) and thenstirred for 1 day at room temperature. The solution was concentrated andthe residue was distributed between ethyl acetate and 2N citric acid,the ethyl acetate phase was washed with water, dried with sodium sulfateand concentrated. Recrystallization was effected from ethyl acetate andpetroleum ether. Yield: 10.45 g (82 percent; melting point: 186 189 C;[01],, 49.5 (c 2, in methanol).

Xl. Further isolated l-hydroxy-benzotriazole ester 1. Z-Gly-OBT 2.1 g ofZ-Gly-OH (10 millimols) and 1.5 g of 1- hydroxy-benzotriazole (11millimols) were reacted in tetrahydrofurane with 2.2 g of DCC asdescribed in Example IV 1. Yield: 1.6 g; melting point: 147 C (fromisopropanol).

2.6g of Z-Thr-OH (l0 millimols), and 1.5 g of 1- hydroxy-benzotriazole(1 1 millimols) were reacted with 2.2 g of DCC in tetrahydrofurane asdescribed in Example 1V 1. Yield: 2.5 g; melting point: 150 153 C (fromisopropanol).

3. Z-AsN(Mbh)-OBT 4.6 g of Z-AsN(Mbh)-OH (10 millimols), and 1.5 g ofl-hydroxy-benzotriazole (ll millimols) were reacted with 2.2 g of DCC inDMF as described in Example IV 1. Yield: 4.0 g; melting point: 158C(boiled up with isopropanol).

4. Z-Phe-( l -hydroxy-5-methyl-benzotriazole)-ester 1 l g of DCC wereadded to a solution cooled to 0 C of 15 g of Z-Phenyl-alanine (50millimols) and 8.25 g of l-hydroxy-5-methyl-benzotriazole (55 millimols)in 150 ml of absolute tetrahydrofurane. The whole was stirred for 1 hourat 0 C and for 1 hour a t room temperature. The precipitate was filteredoff with suction and the filtrate was concentrated. The residue wasrecrystallized from isopropanol. Yield: 10.65 g (50 percent of thetheory); melting point: 153 155 C.

room temperature, and diluted with a small amount of g fdimethylformamide. The precipitate was filtered off bzn'fg iiazblqwairjb 172-3 C,H;1T,.0.5H,0(

with suction and the filtrate was concentrated. The 1741) CQIC-C residuewas boiled up with isopropanol, filtered off with 22:??? :3

suction and washed with ether. Yield: 2.75 g (41 per- 5 5.1,253

cent of the theory); melting point: 170 173 C. (6

X11. Preparation of new l-hydroxy-benzotriazoles methylbenzotriazolesubstituted in the nucleus which may be used as b C1H1N101149J)additives iiifftia General procedures: 10 Found. 56.4, 4.7,

a. 0.1 mol of an aromatic o-chloro-nitro compound (77 was boiled for 5hours under reflux in 50 ml of ethanol l-hydroxy-4- with g of 100percent hydrazine-hydrate (0.3 mol). azg ggs b 151 C H N O (149 I) Thewhole was cooled and the precipitate was filtered 5 Q J 5637" H off withsuction. If nothing precipitated, the mother 4.73,N28.18; liquor wasconcentrated. The precipitate or the residue ggfg was dissolved inwater, and extracted with ether if (71 152.5 necessary. The aqueousphase was acidified with concentrated hydrochloric acid and theprecipitate was filbenzotriazole tered off with suction. The results areshown in Table 4. (611131101) b 193-4 J' b. 0.1 mol of an aromatico-chloro-nitro compound gg gfi 'g fi was heated for 10 hours to 110 C inan autoclave in 50 Found. 58.8, 5.5, ml of alcohol with 15 g of 100percent hydrazine- (60 hydrate (0.3 mol). Working up was effected as 25l-hydroxy-fi-methyl-S- described under (a). The results are shown inTable 4. g fi c. 0.1 mol of an aromatic o-chloro-nitro compound ffiggfijji i b 21643BH'N0 (174.2)CalQC were boiled for 5 hours under refluxin 50 ml of ethanol H N u dwith 27.2 ml of triethylamine 0.2 mol) and 5g of 100 (17%, percent hydrazine-hydrate (0.1 mol). The whole was y qythen concentrated and the residue was worked up as aifig gfiaizgi c 25C H Cm 0 (183 6) described under (a). The results are shown in Table 4.2 1513, i T a b 1 e 4 p 3.30, N 22.39; New l-hydroxy-benzotriazolessubstituted at the V (37 1 gga 1111616118 6-ch1oro-1-hydroxy-5-isopropylbenzotriazole Compounds (recrystal- Method, Melting Analysis(ethanol/water) c 173-4 C,l-1 ,C1N,O (211.7)

I h dmx 6 lized) point 511608572 tryifluoryvmethyl- Found. 50:8, 4.7,benzotriazole 4 w 7 g I W 201 (water) a 148 9 w clam, l

|,98,N20 69; 1. In the method of synthesizing a peptide which g gcomprises reacting: (A) a first reagent which is (1) an (90 a-aminoacid, in its D- or L-form, found in naturallyy y occurring peptides, (2)B-alanine, or (3) a peptide built :ssgmgzgga up from these amino acids,said first reagent having a amide (water) a 214 1 s 4 a free carboxygroup, all other functional groups thereof gg gfiggg i which requireprotection being protected; with Found. 36.5, 3.8, (B) a second reagentwhich is (1) an a-ammo acid, in its D -or L-form, found innaturally-occurring pepbenzotriazoie, tides, (2) B-alanine, or (3) apeptide built up from these amino acids, said second reagent having agif g ggfr li a cwHuNloas (2703) free amino group, all other functionalgroups Ca1c.C44.45,H5 thereof which require protection being protected;

1 (C) a third reagent which is dicyclohexylcarbodiil-hydroxy -fimide,diisopropylcarbodiimide, or a water-soluble gggggxgf carbodiimide; in anorganic solvent at a tempera- (aicohoi 50 a 071-19140, "420 ture from-20C. to 40C., the improvement wherein said reagents are reacted insubstantially "fi gg'gg equivalent amounts in the presence of one to tworoll-1.14217, 1.0, equivalents of l hydmxy s 27-4/29-8 v (D) al-hydroxy-benzotriazole of the formula .iiieni u1 MWybenzotriazole(ethanol)b 215 CH ,N 0, .1 N

Ca1c.C 50.90, H Rn l 4.27, N 25.45;

Found. 50.9, 4.2, 25.7

wherein R is at least one member selected from the group consisting ofhalogen, trifluoromethyl, nitro,

found in naturally-occurring peptides, (2) B-alanine, or

(3) a peptide built up from these amino acids, said first reagent havingafree carboxy group, all other functional groups thereof which requireprotection being protected; with one equivalent part of (B)dicyclohexyl-carbodiimide, diisopropylcarbodiimide, or a watersolublecarbodiimide; and one to two equivalent parts of (C) al-hydroxyj-benzotriazole of the formula Rn LL wherein R is at least onemember selected from the group consisting of halogen, trifluoromethyl,nitro, sulfamoyl, carbamoyl, cyano, lower alkyl, methoxy,

and ethoxy, and n is 0 or an integer from 1 to 4; in an organic solventat C. to 40C. to form an ester of I r reactive-interi said,l-hydroxy-benzotriazole as a mediate, and then'further reacting saidreactive intermediate, in an organic solvent at a temperature from I20C. to 40C. with one equivalent part of a further reagent (D) which is(1) an a-amino acid, in its D- or L-form, found in naturallyoccurringpeptides, (2) B- alanine, or (3) a peptide built up from. these aminoacids, said further reagent having a free'amino group, all otherfunctional groups thereof which require protection being protected.

4. A method as in claim 3 wherein said carbodiimide I isdicyclohexylcarbodiimide.

5. A method as in claim 3 wherein said reactiveintermediate is isolatedprior to reaction with said further I reagent (D).

2. A method as in claim 1 wherein said carbodiimide isdi-cyclohexylcarbodiimide.
 3. The method of synthesizing a peptide whichcomprises reacting one equivalent part of (A) a first reagent which is(1) an Alpha -amino acid, in its D- or L-form, found innaturally-occurring peptides, (2) Beta -alanine, or (3) a peptide builtup from these amino acids, said first reagent having a free carboxygroup, all other functional groups thereof which require protectionbeing protected; with one equivalent part of (B)dicyclohexyl-carbodiimide, diisopropylcarbodiimide, or a water-solublecarbodiimide; and one to two equivalent parts of (C) a1-hydroxy-benzotriazole of the formula
 4. A method as in claim 3 whereinsaid carbodiimide is dicyclohexylcarbodiimide.
 5. A method as in claim 3wherein said reactive inter-mediate is isolated prior to reaction withsaid further reagent (D).